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Background: Neurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. Alpha-lipoic acid (LA) has been used as a therapeutic approach for a variety of neural disorders. We recently reported that LA prevents local anesthetics-induced neurite loss. In this study, we hypothesized that LA administration promotes neurite outgrowth. Methods: To test our hypothesis, we treated mouse neuroblastoma N2a cells and primary neurons with LA. Neurite outgrowth was evaluated by examination of morphological changes and by immunocytochemistry for α-tubulin-3. ROS production was examined, as were the phosphorylation levels of ERK and Akt. In separate experiments, we determined the effects of the inhibition of ERK or PI3K/Akt as well as ROS production on LA-induced neurite outgrowth. Results: LA promoted significantly neurite outgrowth in a time- and concentration-dependent manner. LA stimulation significantly increased the phosphorylation levels of both Akt and ERK and transiently induced ROS production. PI3K/Akt inhibition did not affect LA-induced neurite outgrowth. However, the inhibition of ERK activation completely abolished LA-induced neurite outgrowth. Importantly, the prevention of ROS production by antioxidants attenuated LA-stimulated ERK activation and completely abolished LA-promoted neurite outgrowth. Conclusion: Our data suggest that LA stimulates neurite outgrowth through the activation of ERK signaling, an effect mediated through a ROS-dependent mechanism. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Lithium, a drug used for the treatment of bipolar disorder, has been shown to affect different aspects of neuronal development such as neuritogenesis, neurogenesis and survival. The underlying mechanism responsible for lithium's influence on neuronal development, however, still remains to be elucidated. In the present study, we demonstrate that lithium increases the phosphorylation of extracellular-signal regulated kinases (ERKs) and protein kinase B (Akt) and promotes neurite outgrowth in mouse N2a neuroblastoma cells (N2a). The inactivation of mitogen-activated protein kinase kinase (MEK)/ERKs signaling with a MEK inhibitor inhibits neurite outgrowth, but it enhances Akt activation in lithium-treated N2a cells. Furthermore, the inactivation of phosphoinositide-3-kinase (PI3K)/Akt signaling with a PI3K inhibitor increases both lithium-induced ERKs activation and lithium-induced neurite outgrowth. Taken together, our study suggests that lithium-induced neurite outgrowth in N2a cells is regulated by cross-talk between the MEK/ERKs and PI3K/Akt pathways and requires the activation of the MEK/ERKs signaling.

Despite over 60 years of study, the molecular pathways and mechanisms governing limb outgrowth and patterning remain poorly understood. Fgfs expressed in the AER are known to be necessary and sufficient for proximodistal limb outgrowth and have been proposed to have a chemoattractive role. Wnt5a is a secreted factor which is expressed in a gradient in the distal limb with the highest concentration next to the AER. The presence of the AER is necessary to establish this gradient. Expression of Wnt5a in a concentration dependant manner can be induced in the limb through the implantation of a bead soaked in recombinant Fgf4 protein. This indicates that Fgfs from the AER may establish the gradient of Wnt5a in the limb mesenchyme. Wnt5a-/- mutants exhibit severe shortening of the face, limbs, and body axis, with limbs being progressively truncated proximally to distally. In normal limb proximodistal outgrowth, cells are seen to grow directionally toward the AER. Previous studies done in the Barrow lab, as well as those done by myself, have shown that if a portion of the AER is removed and the cells proximal to this area are labeled, those which are close enough to intact AER will redirect their growth toward this intact AER. When Wnt5a secreting cells are implanted in the limb mesenchyme of the chick this ectopic source of Wnt5a is sufficient to redirect the growth of the mesenchyme cells toward the Wnt5a source. This indicates that the AER may mediate directed growth of limb mesenchyme cells through the establishment of the Wnt5a gradient which provides positional information to the cells. This Wnt5a gradient results in the recruitment of the mesenchyme cells toward the AER. The Ror2 receptor has been found to be involved in several different pathways involving Wnt5a which are involved in changes in polarity and migration. This makes Ror2 a likely candidate for causing changes in cell polarity and migration during distal outgrowth in the limb. To test whether Ror2 is necessary for the polarizing response of limb mesenchyme cells to the Wnt5a gradient in vivo I co-transfected a dominant-negative Ror2 (Ror2ΔC) and a GFP expression vector in the embryonic chick limb using sonoporation. Limb mesenchyme cells transfected with dominant-negative Ror2 grew as radial clones in contrast to the directional outgrowth of the control limb mesenchyme cells along the proximodistal axis. This indicates that cells expressing the dominant-negative Ror2 could no longer respond to the Wnt5a gradient in the limb mesenchyme. This supports a role for Ror2 as a receptor or co-receptor for Wnt5a in mediating directional growth and movement during proximodistal outgrowth and patterning in the limb.

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The mig-10 gene in C. elegans is required for proper axon guidance and/or cell migration of certain neurons during development. In mig-10 (ct41) mutant worms, there is incomplete migration of the anterior lateral microtubule cells (ALMs), hermaphrodite specific neuron (HSN), left coelomocyte cells (ccL), and canal associated neuron (CAN) (Manser and Wood, 1990). The mig-10 (ct41) mutation also causes axon guidance defects in the IL2 neurons, and it enhances unc-6 defects in the axon guidance of the anterior ventral microtubule cell (AVM) (Rusiecki, 1999; C. Quinn, personal communication). mig-10's function in axon guidance and neuronal migration is unknown, but is believed to be involved in a signal transduction pathway that uses a G-protein, such as ras. The two mig-10 transcripts discussed in this thesis, mig-10 A and mig-10 B, encode proteins that are similar to Grb-7 and Grb-10 proteins, which are also believed to function in a signal transduction pathway (Manser et al., 1997). One of these similarities is the presence of a proline-rich region, which may be used to bind another protein (Manser et al., 1997). The MIG-10 A protein has an additional proline region, compared to MIG-10 B, which may indicate that the MIG-10 A and B proteins are utilized in different cells, or at different developmental stages. As a first step in learning where MIG-10 is expressed, mig-10 (ct41) mutant worms containing a wild-type mig-10 B::GFP fusion were constructed. Rescue of the mutant phenotype would indicate that the expression pattern of the transgene was similar to that of the endogenous gene. As this experiment did not allow for rescue, even after integration of the construct, a strain of worms containing a mig-10 promoter::GFP transgene was used. Preliminary observations of this strain indicated that mig-10 is expressed in neuronal tissue. The AIY neurons were observed in wild-type and mig-10 (ct41) worms to determine if they are affected by the mig-10 mutation as previously reported (O. Hobert, personal communication). As no difference was detected, the AIYs were not used in any further experiments. In order to determine which cells require functional MIG-10 protein for the proper development/migration of neurons to occur, mig-10 (ct41) worms containing mec-3 promoter::mig-10 A or B cDNA transgenes were constructed. The mec-3 promoter drives expression of the mig-10 cDNA in the ALM neurons and other touch cells early in the development of the embryo. If these transgenes rescued the ALM migration defect, then mig-10 would be acting cell autonomously in ALM. Partial rescue was obtained, which may be due to the need for both of the mig-10 transcripts to be expressed in the same cell; alternatively, one or both transcripts may need to be expressed in a cell nonautonomous fashion in addition to being expressed cell autonomously. Low production of the rescuing protein, or expression of the protein at a later developmental stage than is needed for rescue to occur, may also have been the cause of the partial rescue. Future work in this area includes putting mig-10 promoter::mig-10 A or B cDNA in mig-10 (ct41) background to investigate if the different transcripts rescue different aspects of the mig-10 phenotype. The mig-10 A and mig-10 B cDNA constructs could also be expressed in the same worm in an attempt to correct for partial rescue that may be due to the lack of both MIG-10 proteins.

Les troubles du spectre autistique (TSA) affectent un individu sur 100 et sont caractérisés par des déficits de la communication et des interactions sociales, et par des comportements restreints et répétitifs. Les TSA présentent une forte composante génétique ; les premiers gènes impliqués ont été identifiés au laboratoire et codent des protéines d’adhérence ou d’échafaudage localisées à la synapse : les neuroligines (NLGN) et SHANK. Nous nous sommes intéressés à l’implication dans les TSA des contactines (CNTN), un groupe de six molécules d’adhérence neurales de la superfamille des immunoglobulines. Ces protéines sont ancrées à la membrane plasmique par un groupement glycosyl phosphatidylinositol (GPI) et peuvent être sécrétées par clivage du GPI. Elles interviennent dans des processus variés du développement neuronal comme la croissance neuritique, le guidage et la fasciculation des axones ou la myélinisation. Des études génétiques ont suggéré l’implication des contactines 4 à 6 dans les TSA, mais aucune étude fonctionnelle n’a confirmé cette hypothèse. Ce travail de thèse associe une analyse génétique, une analyse fonctionnelle sur des cultures de neurones primaires de rat et une analyse structurale par modélisation moléculaire. Nous avons identifié plusieurs "Copy-Number Variants" (CNV) dans les gènes CNTN (essentiellement des délétions affectant CNTN5 et CNTN6) et observé une tendance à l’enrichissement chez les patients par rapport aux individus témoins. Le séquençage des exons codants de CNTN5 et CNTN6 chez plus de 200 patients et 200 témoins nous a ensuite permis d’identifier des variants ponctuels non synonymes. Les variants privés (présents chez un seul individu ou dans une seule famille) sont plus fréquents chez les patients que chez les témoins. Les CNV et les variants ponctuels sont hérités, de parents pour la plupart non atteints, ce qui suggère que les altérations des contactines constituent des facteurs de vulnérabilité aux TSA plutôt que des facteurs causaux. Afin de déterminer l’effet fonctionnel des variants ponctuels rares, nous avons comparé l’effet sur la neuritogenèse des CNTN mutées à celui des CNTN sauvages. Nous avons ainsi analysé, sur plusieurs centaines de neurones par condition, la longueur et la ramification des neurites dans un système de co-culture avec des cellules HEK surexprimant la CNTN. La plupart des protéines CNTN5 et CNTN6 mutées présentent des effets différents de ceux des protéines sauvages (inhibition ou augmentation des effets positifs de celles-ci). Le dernier objectif de cette étude consistait à évaluer l’influence de certains de ces variants sur l’interaction des CNTN, via les domaines immunoglobuline (Ig) 2 et 3, avec l’un de leurs ligands, le récepteur à activité tyrosine phosphatase PTPRG. Par homologie avec la structure cristallographique déjà résolue pour les quatre premiers domaines Ig de CNTN4 de souris, nous avons modélisé cette région pour les CNTN5 et 6 humaines, sauvages et mutées. Nous avons ainsi pu prédire que certains variants étaient susceptibles de modifier les liaisons ioniques ou l’encombrement stérique dans cette région d’interaction. L’ensemble de nos résultats démontre l’existence d’effets fonctionnels délétères de plusieurs variants rares des contactines retrouvés chez les patients atteints de TSA. La présence de ces variants rares chez des apparentés non atteints indique que les altérations des CNTN s’inscrivent dans un modèle de "multiple hit", qui propose que l’autisme puisse résulter de la combinaison de plusieurs atteintes génétiques, chacune représentant un facteur de risque à effet modéré et n’entraînant pas, à elle seule, le développement du trouble. Le séquençage d’exomes et de génomes entiers, en cours au laboratoire, permettra une meilleure compréhension de ces atteintes génétiques multiples. / Autism Spectrum Disorders (ASDs) affect one individual out of 100 and are characterised by deficits in communication and social interactions, and by restricted and repetitive behaviours. ASDs display a strong genetic component ; the first genes involved were identified in our laboratory and encode for cell-adhesion or scaffolding proteins localised at the synapse : neuroligins (NLGNs) and SHANKs. We were interested in the implication, in the ASDs, of contactins (CNTNs), a group of six neural cell-adhesion molecules of the immunoglobulin superfamily. These proteins are anchored to the plasma membrane by a glycosyl phosphatidylinositol (GPI) and can be secreted by cleavage of this anchor. They participate in various processes of neuronal development such as neurite outgrowth, axon guidance and fasciculation, and myelination. Genetic studies have suggested the involvement of contactins 4, 5 and 6 in the ASDs, but no functional study has confirmed this hypothesis. The present work combines a genetic analysis, a functional analysis on cultured primary rat cortical neurons and a structural analysis by molecular modelling. We identified several "Copy-Number Variants" (CNVs) in CNTN genes (mainly deletions affecting CNTN5 and CNTN6) and observed a trend of enrichment in patients compared to control individuals. Subsequent sequencing of CNTN5 and CNTN6 coding exons in more than 200 patients and 200 controls allowed us to identify non synonymous single-nucleotide variants (SNVs). Private variants (present only in one individual or one family) are enriched in patients compared to controls. CNVs and SNVs are inherited, mainly from unaffected parents, which suggests that impairments in contactins represent susceptibility factors for ASDs, rather than causal factors. In order to determine the functional effects of rare SNVs, we compared the effect on neuritogenesis of mutant CNTNs to that of WT CNTNs. We therefore analysed, on several hundreds of neurons per condition, the length and branching of neurites in a co-culture system with HEK cells overexpressing a CNTN protein. Most of CNTN5 and CNTN6 mutant proteins either inhibited or increased the positive effects of WT proteins. The last aim of the present study consisted in evaluating the influence of some of these variants on the interaction of CNTNs, via their immunoglobulin (Ig) domains 2 and 3, with one of their ligands, the protein tyrosine phosphatase receptor PTPRG. Using homology with the crystal structure that had already been solved for the first four Ig domains Ig of mouse CNTN4, we modelled this region for human CNTN5 and CNTN6, WT and mutated. We have thus been able to predict that some variants were likely to alter ionic bonds or steric constraints in this interaction module. Taken as a whole, our results demonstrate that several rare CNTN variants found in patients with ASD have deleterious functional effects. The presence of these rare variants in unaffected relatives indicates that CNTN impairments fit into a "multiple hit" model, according to which autism may result from the combination of several genetic defects, each being a risk factor with moderate effect but not triggering, in itself, the development of the disorder. Sequencing of exomes and whole genomes, ongoing in the laboratory, will allow better understanding of those multiple genetic impairments.